(1) Field of the Invention
The present invention relates to novel alkoxylactones, alkoxylactams and alkoxythiolactams, to methods for controlling processes based on microbial interaction using said alkoxylactones, alkoxylactams and alkoxythiolactams, to uses of said alkoxylactones, alkoxylactams and alkoxythiolactams, and to agents containing said alkoxylactones, alkoxylactams and alkoxythiolactams.
Microorganisms communicate with each other by means of a plurality of different signals. In particular, the mechanisms of communication and interaction of bacteria are extensively described in the prior art.
Specific histidine kinases or homologous proteins that are intended for use in screening for antibacterial substances are described in the documents WO 00/56154; EP 0881297; U.S. Pat. No. 6,287,836; EP 0909820 and U.S. Pat. No. 6,001,600. The kinases derive exclusively from gram-positive organisms.
In WO 01/49708, a peptide with at least 6 but less than 200 amino acids is used for blocking a response regulator. However, no signal molecules are blocked, rather the activation of the next protein in the signal chain is inhibited.
The formation of mature biofilms by or with bacteria is dependent on the communication between bacterial cells over various extracellular signal substances.
Depending on the cell count density of the adhering bacteria, any level of this messenger substance above a minimum concentration effects an activation of the biofilm formation as well as inducing additional virulent genes. This phenomenon is called “Quorum Sensing” and fundamentally opens up the possibility of controlling biofilms, without destroying the living germs in the biofilm.
(2) Description of Related Art, Including Information Disclosed Under 37 C.F.R. §§1.97 and 1.98.
Up to now, a great many signal molecules have been identified in gram negative and gram-positive germs [Miller, M. B., Bassler, B. L. (2001) Quorum sensing in bacteria. Annu Rev Microbiol. 55, 165-199; Kleerebezem, M., Quadri, L. E. (2001) Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior. Peptides 22, 1579-1596].
The structure of these signal molecules has been particularly well investigated for gram-negative organisms. The signal substances (pheromones) found in these organisms are often members of a group of different N-acyl-L-homoserine lactones (AHL) that differ in the length of the N-acyl side groups and by modifications at the C3 position (3-oxo or 3-hydroxy groups) [Greenberg, E. P. (1997) Quorum sensing in Gram-negative bacteria. ASM News 63, 371-377].
The mechanisms are not yet as well understood for gram-positive bacteria. Often, smaller peptides are described as the signal molecules. [Kleerebezem, M., Quadri, L. E. (2001) Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior. Peptides 22, 1579-1596].
However, the fundamental principle is similar. Each signal molecule is recognized and bound by specific, generally membrane-bound cellular receptors (two component systems).
In gram-negative bacteria, specific histidine kinases inter alia act as the implied receptors. These histidine kinases are constituents of the so-called two-component system. The second component is formed by the so-called response regulator. When a signal molecule binds to such a histidine kinase, the response regulator is activated by it. The activated response regulator acts itself as the activator of many different cellular processes [Chang C, Stewart, The two-component system. Regulation of diverse signaling pathways in prokaryotes and eukaryotes. Plant Physiol. 1998 Jul; 117(3):723-31.]
Whereas there are a large number of such two-component systems with the most varied functions in all cells, intracellular transcription-activator-proteins (“LuxR-proteins”) are mainly responsible for the biofilm development by gram-negative bacteria. Due to their functionality, the proteins of the LuxR class are firstly able to bind AHLs, but secondly also to undergo interactions with regulation areas of DNA molecules. [Eberl L, N-acyl homoserinelactone-mediated gene regulation in gram-negative bacteria, Syst Appl Microbiol. 1999 Dec; 22(4):493-506; Michael B, Smith J N, Swift S, Heffron F, Ahmer B M. SdiA of Salmonella enterica is a LuxR homolog that detects mixed microbial communities. J Bacteriol. 2001 Oct; 183(19):5733-42; Gray K M, Garey J R., The evolution of bacterial LuxI and LuxR quorum sensing Microbiology. 2001 Aug; 147(Pt 8):2379-87.].
Recently, it is known that only the N-terminal part of the transcription activator protein (ca. 200 amino acids) is responsible for binding the AHL molecule. [Zhang R G, Pappas T, Brace J L, Miller P C, Oulmassov T, Molyneaux J M, Anderson J C, Bashkin J K, Winans S C, A., Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA. Nature. 2002 Jun 27; 417(6892):971-4.].
From the prior art and science, essentially three different concepts are described for biofilm prevention or control.
1. Cleavage of the Messenger Substances by Enzymes, e.g., Lactonases.
Recently, various enzymes have been isolated from gram-positive bacillae, which specifically cleave the AHL molecule and can thereby switch off these signal paths (“Quorum quenching”). These lactonases have been described many times [Dong Y H, Wang L H, Xu J L, Zhang H B, Zhang X F, Zhang L H. (2001) Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature. 411, 813-817; WO 0185664; WO 0216623].
2. Blocking/Binding of Messenger Substances per se, e.g., with Antibodies.
A further possibility to prevent biofilms is the use of AHL-specific antibodies. By binding an antibody of this type to the messenger substance, the latter is no longer able to bind to its original receptor (=histidine kinase) [WO 0194543]. However, due to the high costs to manufacture the antibody, this method is at most feasible for medical purposes.
3. Blocking the Cellular Receptors of the Messenger Substances, e.g., by Means of Structurally Analogous Substances.
Furanones and furanone derivatives are particularly known as structurally analogous substances. [Manefield M, de Nys R, Kumar N, Read R, Givskov M, Steinberg P, Kjelleberg. Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression displacing the AHL signal from its receptor protein. Microbiology. 1999 Feb; 145 (Pt 2):283-91; WO 9629392; WO 0168091; WO 0168090; WO 0176594].
Furanones illustrate AHL analogues in terms of their structure. They interrupt the signal transmission path, in that they competitively suppress the AHL signal substances and bind to the intracellular target molecule (intracellular transcription activator protein). The growth of the affected germs is not inhibited at low concentrations by the effect of the furanone.
Besides the addition of furanones, the use of antibodies or antibody fragments has also been discussed in individual cases. [Bryers, J. D., 2001, Gene therapy approach to preventing bacterial colonization of biomaterials, Abstracts of papers, 222nd ACS National Meeting].
The present invention is based on the problem of providing novel substances by means of which the interaction of microbes with one another can be controlled and regulated.